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Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

  • Jatinder S. Josan
  • Channa R. De Silva
  • Byunghee Yoo
  • Ronald M. Lynch
  • Mark D. Pagel
  • Josef Vagner
  • Victor J. HrubyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 716)

Abstract

The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening ­studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents.

Key words

Cy5 DELFIA DTPA DOTA Dual-modal agents Heteromultivalent ligands,Lanthaligands PARACEST MRI Solid-phase synthesis Targeted imaging agents 

Abbreviations

δ-OR

Delta-opioid receptor

Ado

8-Amino-3,6-dioxaoctanoyl

Arsenazo (III)

2,7-Bis(2-arsenophenylazo)-1,8-dihydroxyaphthalene-3,6-disulfonic acid

BB

Bromophenol blue

Boc

tert-Butyloxycarbonyl

CCK(6)

Nle-Gly-Trp-Nle-Asp-Phe-NH2

CCK(8)

Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2

CCK2R

Cholecystokinin receptor subtype 2

CDI

N,N′-carbonyldiimidazole

CEST

Chemical exchange saturation transfer

CH3CN

Acetonitrile

CT

Computed tomography

Cy5

Cyanine 5 dye

DCM

Dichloromethane

DELFIA

Dissociation-enhanced lanthanide fluoroimmunoassay

DIC

N,N′-diisopropylcarbodiimide

DIEA

Diisopropylethylamine

DMBA

1,3-Dimethylbarbituric acid

DMEM

Dulbecco’s modified eagle medium

DMF

N,N′-dimethylformamide

DMSO

Dimethylsulfoxide

Dmt

2′,6′-Dimethyl-l-tyrosine

DOTA

1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid

DPLCE

c[dPen2,Cys5]enkephalin

DTPA

Diethylenetriamine-N,N,N′,N′,N²-pentaacetic acid

EDC

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride

EDT

1,2-Ethanedithiol

ESI-MS

Electrospray ionization-mass spectrometry

Fmoc

(9H-fluoren-9-ylmethoxy)carbonyl

FT-ICR

Fourier transform-ion cyclotron resonance

HBTU

2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro-phosphate

hMC4R

Human melanocortin-4 receptor

HOBt

N-hydroxybenzotriazole

HOCt

6-Chloro-1H-hydroxybenzotriazole

htBVLs

Heterobivalent ligands

htMVL

Heteromultivalent ligand

MALDI-TOF

Matrix-assisted laser desorption ionization-time of flight

MRI

Magnetic resonance imaging

MSH

Melanocyte-stimulating hormone

MSH-7

Ser-Nle-Glu-His-dPhe-Arg-Trp

Mtt

4-Methyltrityl

NDP-α-MSH

Ac-Ser-Tyr-Ser-Nle-Glu-His-dPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2

NHS

N-hydroxysuccinimide ester

NIR

Near-infrared

PARACEST

Paramagnetic chemical exchange saturation transfer

Pbf

2,2,4,6,7-Pentamethyl-dihydrobenzofuran-5-sulfonyl

PEG

Polyethyleneglycol

Pego

19-Amino-5-oxo-3,10,13,16-tetraoxa-6-azanonadecan-1-oic acid

RP-HPLC

Reverse-phase high-performance liquid chromatography

SPECT

Single photon emission computed tomography

SPPS

Solid-phase peptide synthesis

TA

Thioanisole

tBu

tert-butyl

TFA

Trifluoroacetic acid

THF

Tetrahydrofuran

Tic

1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid

TIS

Triisopropylsilane

TRL

Time-resolved luminescence

Trt

Triphenylmethyl (trityl)

Notes

Acknowledgments

The authors would like to thank Prof. Robert J. Gillies and his team for development of various cell lines and animal model for δOR tumor animal model described in this work. This work was supported by the National Cancer Institute through NIH Grant R21CA133455-01, R01 CA09736, and R01 CA 123547, and by the U.S. Army Medical Research and Materiel Command under W81XWH-04-1-0731. This work was also supported by the Northeastern Ohio Animal Imaging Resource Center, an NIH-funded program, R24CA110943.

References

  1. 1.
    Josan, J. S., Vagner, J., Handl, H. L., Sankaranarayanan, R., Gillies, R. J., and Hruby, V. J. (2008) Solid-phase synthesis of heterobivalent ligands targeted to melanocortin and cholecystokinin receptors. Int J Pep Res Ther. 14: 293–300.CrossRefGoogle Scholar
  2. 2.
    Vagner, J., Xu, L., Handl, H., Josan, J. S., Morse, D. L., Mash, E. A., Gillies, R. J., and Hruby, V. J. (2008) Heterobivalent ligands crosslink multiple cell-surface receptors: the human melanocortin-4 and delta-opioid receptors. Angew Chem Int Ed. 47: 1685–1688.CrossRefGoogle Scholar
  3. 3.
    Xu, L., Vagner, J., Josan, J., Lynch, R. M., Morse, D. L., Baggett, B., Han, H., Mash, E. A., Hruby, V. J., and Gillies, R. J. (2009) Enhanced targeting with heterobivalent ligands. Mol Cancer Ther. 8: 2356–2365.PubMedCrossRefGoogle Scholar
  4. 4.
    Sokolov, K., Follen, M., and Richards-Kortum, R. (2002) Optical spectroscopy for detection of neoplasia. Curr Opin Chem Biol. 6: 651–658.PubMedCrossRefGoogle Scholar
  5. 5.
    Ballou, B., Ernst, L. A., and Waggoner, A. S. (2005) Fluorescence imaging of tumors in vivo. Curr Med Chem. 12: 795–805.PubMedCrossRefGoogle Scholar
  6. 6.
    Frangioni, J. V. (2003) In vivo near-infrared fluorescence imaging. Curr Opin Chem Biol. 7: 626–634.PubMedCrossRefGoogle Scholar
  7. 7.
    Sevick-Muraca, E. M., Houston, J. P., and Gurfinkel, M. (2002) Fluorescence-enhanced, near infrared diagnostic imaging with contrast agents. Curr Opin Chem Biol. 6: 642–650.PubMedCrossRefGoogle Scholar
  8. 8.
    Mujumdar, R. B., Ernst, L. A., Mujumdar, S. R., Lewis, C. J., and Waggoner, A. S. (1993) Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjug Chem. 4: 105–111.PubMedCrossRefGoogle Scholar
  9. 9.
    Handl, H. L., Vagner, J., Yamamura, H., Hruby, V. J., and Gillies, R. J. (2004) Lanthanide-based time-resolved fluorescence of in cyto ligand–receptor interactions. Anal Biochem. 330: 242–250.PubMedCrossRefGoogle Scholar
  10. 10.
    Handl, H. L., and Gillies, R. J. (2005) Lanthanide-based luminescent assays for ligand-receptor interactions. Life Sci. 77: 361–371.PubMedCrossRefGoogle Scholar
  11. 11.
    Pandya, S., Yu, J., and Parker, D. (2006) Engineering emissive europium and terbium complexes for molecular imaging and sensing. Dalton Trans. 23: 2757–2766.PubMedCrossRefGoogle Scholar
  12. 12.
    Selvin, P. (2002) Principles and biophysical applications of lanthanide-based probes. Annu Rev Biophys Biomol Struct. 31: 275–302.PubMedCrossRefGoogle Scholar
  13. 13.
    Steinkamp, T., and Karst, U. (2004) Detection strategies for bioassays based on luminescent lanthanide complexes and signal amplification. Anal Bioanal Chem. 380: 24–30.PubMedCrossRefGoogle Scholar
  14. 14.
    Parker, D. (2004) Excitement in f block: structure, dynamics and function of nine-coordinate chiral lanthanide complexes in aqueous media. Chem Soc Rev. 33: 156–165.PubMedCrossRefGoogle Scholar
  15. 15.
    Thunus, L., and Lejeune, R. (1999) Overview of transition metal and lanthanide complexes as diagnostic tools. Coord Chem Rev. 184: 125–155.CrossRefGoogle Scholar
  16. 16.
    Josan, J. S., Morse, D. L., Xu, L., Trissal, M., Baggett, B., Davis, P., Vagner, J., Gillies, R. J., and Hruby, V. J. (2009) Solid-phase synthetic strategy and bioevaluation of a Labeled δ-opioid receptor ligand Dmt-Tic-Lys for in vivo imaging. Org Lett. 11: 2479–2482.PubMedCrossRefGoogle Scholar
  17. 17.
    Handl, H. L., Vagner, J., Yamamura, H. I., Hruby, V. J., and Gillies, R. J. (2005) Development of a lanthanide-based assay for detection of receptor–ligand interactions at the δ-opioid receptor. Anal Biochem. 343: 299–307.PubMedCrossRefGoogle Scholar
  18. 18.
    De-Silva, C. R., Vagner, J., Lynch, R., Gillies, R. J., and Hruby, V. J. (2010) Optimization of time-resolved fluorescence assay for detection of Eu-DOTA labeled ligand-receptor interactions. Anal Biochem. 398: 15–23.PubMedCrossRefGoogle Scholar
  19. 19.
    Leon-Rodriguez, L. M. D., and Kovacs, Z. (2008) The synthesis and chelation chemistry of DOTA-peptide conjugates. Bioconjug Chem. 19: 391–402.PubMedCrossRefGoogle Scholar
  20. 20.
    Uusijärvi, H., Bernhardt, P., Rösch, F., Maecke, H. R., and Forssell-Aronsson, E. (2006) Electron- and positron-emitting radiolanthanides for therapy: aspects of dosimetry and production. J Nucl Med. 47: 807–814.PubMedGoogle Scholar
  21. 21.
    Merbach, A. F., and Toth, E. (2001) The chemistry of contrast agents in medical magnetic resonance imaging. Wiley, New York.Google Scholar
  22. 22.
    Zhang, S., Merritt, M., Woessner, D. E., Lenkinski, R. E., and Sherry, A. D. (2003) PARACEST agents: modulating MRI ­contrast via water proton exchange. Acc Chem Res. 36: 783–790.PubMedCrossRefGoogle Scholar
  23. 23.
    Aime, S., Barge, A., Delli Castelli, D., Fedeli, F., Mortillaro, A., Nielsen, F. U., and Terreno, E. (2002) Paramagnetic lanthanide (III) complexes as pH sensitive chemical exchange saturation transfer (CEST) contrast agents for MRI applications. Magn Reson Med. 47: 639–648.PubMedCrossRefGoogle Scholar
  24. 24.
    Yoo, B., Sheth, V., and Pagel, M. D. (2009) An amine-derivatized, DOTA-loaded polymeric support for Fmoc solid phase peptide synthesis. Tet Lett. 50: 4459–4462.CrossRefGoogle Scholar
  25. 25.
    Ali, M. M., Liu, G., Shah, T., Flask, C. A., and Pagel, M. D. (2009) Using two chemical exchange saturation transfer magnetic resonance imaging contrast agents for molecular imaging studies. Acc Chem Res. 42: 915–924.PubMedCrossRefGoogle Scholar
  26. 26.
    Yoo, B., and Pagel, M. D. (2008) An overview of responsive MRI contrast agents for molecular imaging. Front Biosci. 13: 1733–1752.PubMedCrossRefGoogle Scholar
  27. 27.
    Yoo, B., and Pagel, M. D. (2006) A PARACEST MRI contrast agent to detect enzyme activity. J Am Chem Soc. 128: 14032–14033.PubMedCrossRefGoogle Scholar
  28. 28.
    Yoo, B., Raam, M., Rosenblum, R., and Pagel, M. D. (2007) Enzyme-responsive PARACEST MRI contrast agents: a new biomedical imaging approach for studies of the proteasome. Contrast Media Mol Imaging. 2: 189–198.PubMedCrossRefGoogle Scholar
  29. 29.
    Grieco, P., Lavecchia, A., Cai, M., Trivedi, D., Weinberg, D., MacNeil, T., Van der Ploeg, L. H., and Hruby, V. J. (2002) Structure-activity studies of the melanocortin peptides: discovery of potent and selective affinity antagonists for the hMC3 and hMC4 receptors. J Med Chem. 45: 5287–5294.PubMedCrossRefGoogle Scholar
  30. 30.
    Martinez-Zaguilan, R., Tompkins, L. S., Gillies, R. J., and Lynch, R. M. (1999) Simultaneous analysis of intracellular pH and Ca2+ from cell populations. Meth Mol Biol. 114: 287–306.Google Scholar
  31. 31.
    Edwards, W. B., Fields, C. G., Anderson, C. J., Pajeau, T. S., Welch, M. J., and Fields, G. B. (1994) Generally applicable, convenient solid-phase synthesis and receptor affinities of octreotide analogs. J Med Chem. 37: 3749–3757.PubMedCrossRefGoogle Scholar
  32. 32.
    Krchnák, V., Vágner, J., and Lebl, M. (1988) Noninvasive continuous monitoring of solid-phase peptide synthesis by acid-base indicator. Int J Pept Prot Res. 32: 415–416.CrossRefGoogle Scholar
  33. 33.
    Bräse, S., Kirchhoff, J. H., and Köbberling, J. (2003) Palladium-catalysed reactions in solid phase organic synthesis. Tetrahedron. 59: 885–939.CrossRefGoogle Scholar
  34. 34.
    Gomez-Martinez, P., Dessolin, M., Guibé, F., and Albericio, F. (1999) Nα-Alloc temporary protection in solid-phase peptide synthesis. The use of amine–borane complexes as allyl group scavengers. J Chem Soc Perkin Trans. 1: 2871–2874.CrossRefGoogle Scholar
  35. 35.
    Williams, R. M., Aldous, D. J., and Aldous, S. C. (1990) General synthesis of β, γ-alkynylglycine derivatives. J Org Chem. 55: 4657–4663.CrossRefGoogle Scholar
  36. 36.
    Moore, D. A. (2008) Selective trialkylation of cyclen with tert-butyl bromoacetate. Org Synth. 85: 10–14.Google Scholar
  37. 37.
    Ali, M., Yoo, B., and Pagel, M. D. (2009) Tracking the relative in vivo pharmacokinetics of nanoparticles with PARACEST MRI. Mol Pharm. 6: 1409–1416.PubMedCrossRefGoogle Scholar
  38. 38.
    Liu, G., Ali, M. M., Yoo, B., Griswold, M. A., Tkach, J. A., and Pagel, M. D. (2009) PARACEST MRI with improved temporal resolution. Magn Reson Med. 61: 399–408.PubMedCrossRefGoogle Scholar
  39. 39.
    Liu, G., Li, Y., and Pagel, M. D. (2007) Design and characterization of a new irreversible responsive PARACEST MRI contrast agent that detects nitric oxide. Magn Reson Med. 58: 1249–1256.PubMedCrossRefGoogle Scholar
  40. 40.
    Josan, J. S. (2008) Heteromultivalent ligands directed targeting of cell-surface receptors – implications in cancer diagnostics & therapeutics. Ph.D. dissertation. The University of Arizona, Tucson.Google Scholar
  41. 41.
    Dakubu, S. (1992) Method of determining a biological substance involving labelling with a metal chelate. US Patent 5,124,268.Google Scholar
  42. 42.
    Wilkinson, D. (1999) A one-step fluorescent detection method for lipid fingerprints; Eu(TTA)3.2TOPO. Forensic Sci Int. 99: 5–23.PubMedCrossRefGoogle Scholar
  43. 43.
    Albericio, F., Annis, I., Royo, M., and Barany, G. (2000) Preparation and handling of peptides containing methionine and cysteine. In: Chan, W. C., White P. D. (eds) Fmoc solid phase peptide synthesis, 1st ed., Oxford University Press, Oxford, pp 77–114.Google Scholar
  44. 44.
    Annis, I., Hargittai, B., and Barany, G. (1997) Disulfide bond formation in peptides. Meth Enzymol. 289: 198–221.PubMedCrossRefGoogle Scholar
  45. 45.
    Chen, L., Annis, I., and Barany, G. (2001) Disulfide bond formation in peptides. Curr Protocols Prot Sci. 18.16.11–18.16.19.Google Scholar
  46. 46.
    Fields, G. B., Lauer-Fields, J. L., Liu, R., and Barany, G. (1992) Principles and practice of solid phase peptide synthesis. In: Grant, G. A. (ed) Synthetic Peptides: A User’s Guide, 2nd ed., Oxford University Press, Oxford, pp 93–219.Google Scholar
  47. 47.
    Gisin, B. F. (1972) The monitoring of reactions in solid-phase peptide synthesis with picric acid. Anal Chim Acta. 58: 248–249.PubMedCrossRefGoogle Scholar
  48. 48.
    Rowatt, E., and Williams, R. J. (1989) The interaction of cations with the dye Arsenazo III. Biochem J. 259: 295–298.PubMedGoogle Scholar
  49. 49.
    Barge, A., Cravotto, G., Gianolio, E., and Fedeli, F. (2006) How to determine free Gd and free ligand in solution of Gd chelates. A technical note. Contrast Media Mol Imaging. 1: 184–188.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Jatinder S. Josan
    • 1
  • Channa R. De Silva
    • 1
  • Byunghee Yoo
    • 1
  • Ronald M. Lynch
    • 2
  • Mark D. Pagel
    • 1
  • Josef Vagner
    • 2
  • Victor J. Hruby
    • 1
    Email author
  1. 1.Department of ChemistryUniversity of ArizonaTucsonUSA
  2. 2.Bio5 InstituteUniversity of ArizonaTucsonUSA

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